The long-term goal of this research is to understand signaling pathways that regulate programs of innate immune gene expression by controlling the subcellular localization of regulatory proteins. In the fruit fly Drosophila, signal transduction by the cell surface receptor Toll promotes nuclear translocation of transcription factors governing both innate immunity and embryonic patterning. Pathway function in innate immune responses has been widely conserved, with homologous pathways inducing antimicrobial defenses in both plants and mammals. Infection of flies with particular sets of pathogens activates either the Toll or the Imd signaling pathway. The targets of Toll signaling are Dif and Dorsal, which are NF-?B related transcription factors, and the inhibitory protein Cactus, an I?B homolog. The target of the Imd pathway is a third NF-?B protein, Relish, which is activated by a conserved caspase and by the fly counterpart of the IKK complex. The fact that these pathways have been characterized at both the genetic and molecular level and can be assayed in whole fly and cell culture systems makes them particularly amenable to experimental investigation. It is now possible, therefore, to address fundamental questions about the mechanisms for signal transduction, the coordination of Toll and Imd pathway function, the evolution of pathway architecture, and the overall program for regulation of immune responses. The focus of the proposed research will be to acquire and integrate knowledge of signal transduction mechanism into the context of overall regulation and organization of humoral innate immune defenses. In carrying out these studies, we will take advantage of discoveries regarding the orthologous relationships between fly and mammalian Toll pathway components. We will use RNA interference technology and phosphospecific antibodies to identify the physiologically relevant Cactus kinase. We will also carry out in vitro binding assays and site-directed mutagenesis to test a model for conservation of protein-protein interactions in the Toll pathway. Using an approach based on a state-of-the-art method for transgenic studies in Drosophila, we will test hypotheses regarding the role of binding site number in delineating pathway functions. A combination of site-directed mutagenesis and reporter gene studies will be exploited to refine our knowledge of a cis-regulatory site for immune regulation and to provide the basis for identification of the trans-acting factors. Lastly, we will combine functional studies, expression data, and sequence comparisons across twelve Drosophila genomes to develop a predictive model for innate immune gene regulation. Given the conserved nature of the signaling pathways, the results of the proposed research should be of substantial interest with regard to innate immunity pathways and defenses in a broad range of organisms. PUBLIC HEALTH RELEVANCE Humans and other animals rely on innate immune defenses to recognize and respond to infection by a range of microbial pathogens. Furthermore, abnormal function of innate immune systems contributes to a range of human disorders, including arthritis, heart disease, and cancer. By studying the mechanism and regulation of such response pathways, we will therefore obtain knowledge of broad and substantial significance to human health.